<Review>Stereochemical Diversity in Lignan Biosynthesis and Establishment of Norlignan Biosynthetic Pathway

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Title

<Review>Stereochemical Diversity in Lignan Biosynthesis and

Establishment of Norlignan Biosynthetic Pathway

Author(s)

SUZUKI, Shiro

Citation

Wood research : bulletin of the Wood Research Institute Kyoto

University (2002), 89: 52-60

Issue Date

2002-09-30

URL

http://hdl.handle.net/2433/53121

Right

Type

Departmental Bulletin Paper

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publisher

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Stereochemical Diversity in Lignan Biosynthesis

. and Establishment of Norlignan

Biosynthetic Pathway*l

Shiro SUZUKI*2

(Received May 31, 2002)

Keywords: lign~n,.norlignan, biosynthesis, heartwood substance, stereochemistry,Arctium lappa, Anthriscus sylvestris, Asparagus officmalls

(+)-Secoiso-

(-)-Secoiso-lariciresinol lariciresinol

Fig. I. Chemical structures of (+)- and (- )-enantio-mers of matairesinol and secoisolariciresinol. compounds.

Biological activities are often related to stereochemistry of compounds. Lignans are stereochemically peculiar in natural products. In general, lignan molecules are chiral, and one enantiomer predominates or only one enantiomer is present in each lignan sample isolated from plants. Interestingly, however, the predominant enantiomer varies with the plant sources. For example, optically pure, levorotatory (- )-matairesinol (Fig. 1) was isolated from Forsythia intermedia6) , while the optically pure,

dextrorotatory (+)-matairesinol (Fig. 1) was isolated from Wikstroemia sikokiana7) • ( - )-Secoisolariciresinol (Fig. I) from F. intermedia6) and F. koreana8) is optically pure, whereas (- )-secoisolariciresinol isolated from W. sikokiana is not optically pure [45% enantiomer excess (e.e.)]9). Furthermore, (+)-secoisolariciresinol (78% e.e., Fig. 1) was isolated fromA rctium lappapetioles10). These findings strongly suggest that stereochemical control in lignan formation differs among plant species.

Stereochemical difference between lignan and lignin is also of interest. Both are synthesized by one-electron

OH OH OH OH (-)-Matairesinol OH OH (+)-Matairesinol Contents Introduction

Lignans and norlignans are two major classes of wood extractives, accumulating specifically in heartwood composed of only dead cells and occupying the most of the trunk. These secondary metabolites are called "heartwood substances", which are synthesized in parenchyma cells and spread out from the cells to other xylem elements, followed by the death of the cells. This sequence of metabolic events, heartwood formation, was specific to woody plants but not to herbaceous plants. The reason woody plants are long-lived is partly because they accumulate heartwood substances, some of which prevent wood-degrading fungi from rotting.

In addition to the antimicrobial activity, lignans and norlignans have various biological activitiesl-4). Among them, the antitumor lignan, podophyllotoxin, is of special interest, because it is commercially important as a staring material of etoposide and teniposide, which have been used as anticancer drugs in the hospital. However, the large-scaled exploitation of source plants is decreasing the amount of its natural resources5). Therefore, it is necessary to establish the efficient production system of podophyllotoxin by which we do not need to depend on the small natural resources. The studies on biosynthesis of lignans and norlignans would afford the essential knowledge for biotechnological production of these *1 This article is the abstract of Ph. D. thesis by the author

(Kyoto University, 2002).

*2 Laboratory of Biochemical Control. Introduction

Chapter I Stereochemical diversity in lignan biosynthesis 1-1 Stereochemical diversity in lignan biosynthesis of

Arctium lappa L.

1-2 Stereochemistry of lignan formation in Anthriscus sylvestris (L.) HofTm.

Chapter II Establishment of norlignan biosynthetic pa-thway

II-I Pathway of norlignan biosynthesis

11-2 First enzymatic formation of the norlignan Conclusions

Acknowledgement References

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SUZUKI: Biosynthesis of Lignans and Norlignans Coniferyl alcohol HO Pinoresinol b HO

H3CO~~

OH HO ~ OH b

---.-

I

~ ~ OCH3 OH Secoisolariciresinol LariciresinoI c OH

::::~:

0 d ...." 0

I "

~ OCH3 OH Matairesinol Arctigenin a dirigent proteinllaccase/HzOz b pinoresinolllariciresinol reductase/NADPH c secoisolariciresinol dehydrogenase/NADP dO-methyl transferase/SAM

Fig. 2. Lignan biosynthetic pathway from coniferyl alcohol to arctigenin.

oxidative coupling of hydroxycinnamyl alcohols, but fundamentally differ in optical activity; lignans are optically active, whereas lignins are inactive. These results suggest that lignan biosynthesis involves stereochemically different process from that of lignin biosyn thesis.

Because of these important features, biosynthesis of lignans and norlignans has been receiving widespread interest. In this review, the author describes the recent findings on stereoch~micaldiversity in lignan biosynthesis and the establishment of norlignan biosynthetic pathway.

the presence of NADpI5).

On the other hand, the recent studies in the author's laboratory have revealed that the stereochemistry oflignan biosynthesis varies with plant species. In contrast to cell-free extracts from Forsythia plantsl l), .Umezawa and

Shimada10) isolated (+)-secoisolariciresinol (78% e.e.)

from A. lappa petioles and the cell-free extracts catalyzed the enantioselective formation of (+)-secoisolariciresinol from coniferyl alcohol in the presence of H 20 2 and NADPH. This result indicates that A. lappa has a different stereochemical control in lignan biosynthesis from that of Forsythia plants.

In this chapter, the stereochemistry of lignan formation in two plant species other than Forsythia, e.g. Arctium lappa

and Anthriscus sylvestris, is discussed. First, the author describes thatthe stereochemistry oflignan biosynthesis in

A. lappa is regulated organ-specificallyI6,17) In contrast to the case of A. lappa petioles, (- )-secoisolariciresinol (65% e.e.) was isolated from seeds, and the enzyme preparations from ripening seeds catalyzed the formation of (- )-secoisolariciresinol (38% e.e.) from coniferyl alcohol in the presence of NADPH and H 20 2. In addition, ripening seed enzyme preparation mediated the selective formation of the optically pure (>99% e.e.) (-)-enantiomer of matairesinol from (±)-secoisolariciresinols in the presence of NADP. Second, the author describes . the stereochemistry of lignan formation in A. sylvestris, a herbaceous plant known to produce podophyllotoxin congeners and the same lignans as often found in conifer heartwood (=heartwood lignans,e.g.yatein, hinokinin) 18). The author shows the formation of(+)-lariciresinol (93% e.e.) and ( - )-secoisolariciresinol (95% e.e.) from

(±)-pinoresinols in the presence of NADPH. This result indicates that the stereochemical property of reduction catalyzed byA. sylvestrispinoresinol/lariciresinol reductase (PLR) is similar to those ofForsythiaPLR andA. lappaseed lignan

in diversity Chapter I Stereochemical

biosynthesis

The first enzymatic and enantioselective formation of an optically pure lignan, ( - )-secoisolariciresinol, from achiral coniferyl alcohol with cell-free extracts from

Forsythia intermedia in the presence of H 20 2 and NAD(P)H was reported by U mezawaet al.II) They also demons tra ted the selective oxidation of (- )-secoisolariciresinol to optically pure (- )-matairesinol III the presence of

NAD(P)6).

Lewis and co-workers continued to investigate the processes of secoisolariciresinol and matairesinol formation in Forsythia and established the lignan biosynthetic pathway (Fig. 2). Each step, except for the final conversion from matairesinol to arctigenin 12), is well controlled in terms of stereochemistry; (+)-pinoresinol is formed enantioselectively from achiral coniferyl alcohol with oxidase/oxidan t in the presence of dirigen t protein 13) . The formed (+)-pinoresinol is transformed to (+)-lariciresinol and (- )-secoiso(+)-lariciresinol with pinoresinol! lariciresinol reductase in the presence of NADPHI4), and (- )-secoisolariciresinol was in turn oxidized to (-)-matairesinol with secoisolariciresinol dehydorogenase in

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-PLR.

1-1 Stereochemical diversity in lignan biosynthesis ofArctium lappa L.

Since Shinoda. and Kawagoye isolated arctiin, a glycoside of arctigenin, from seeds ofArctium lappain 1929, seeds of Arctium spp. have been well-known to contain significant amounts of lignansI9-23). Arctigenin isolated from the seed was levorotatory, but the optical purity has not been determined. Suzuki et al.I7) determined the optical purity of lignans (- )-matairesinol and (-)-arctigenin by chiral high-performance liquid chromatography (HPLC) after treating MeOH extracts from the seeds with f3-g1ucosidase. Both lignans' were optically pure (>99% e.e.). This is in good accordance with previous reports; all the dibenzylbutyrolactone lignans of which enantiomeric compositions have so far been determined precisely by chiral HPLC are optically pure24)

In addition, small amounts of secoisolariciresinol were isolated from seeds. In contrast to (

+

)-seoisolariciresinol (78% e.e.) isolated from the petiolesIO), ( ) -secoisolariciresinol (65% e.e.) was isolated from MeOH extract of the seeds after glucosidase treatmentI6). Acid hydrolysis (H2S04 ) also yielded (-~-secoisolariciresinol

(82% e.e.) from the MeOH extractsI ). This is the first

example that different enantiomers of a particular lignan occur predominantly in different organs of a single plant species, indicating the·· stereochemical diversity of lignan biosynthetic mechanism in A. lappa.

U mezawa and Shimada10)reported that the incubation of coniferyl alcohol with the petiole enzyme preparation

gave the (+)-enantiomer of secoisolariciresinol (ca. 20% e.e.) by liquid-chromatography mass spectrometry. However, because of the incomplete separation in the liquid-chromatography, the value was not accurate. Suzuki et al.I7)determined the enantiomeric compositions of lignans, pinoresinol, lariciresinol and secoisolari-ciresinol, by gas-chromatography mass spectrometry after chiral HPLC separation; (+)-pinoresinol (33% e.e.), (+)-lariciresinol (30% e.e.) and (+)-secoisolariciresinol (20% e.e.) were formed with the petiole enzyme from coniferyl alcohol. On the other hand, seed enzyme prepared from A. lappa ripening seeds catalyzed the formation of (- secoisolariciresinol (38 % e.e.), ( - )-pinoresinol (22% e.e.), and (- )-lariciresinol (>99% e.e.) from coniferyl alcohol in the presence of NADPH and H20 2

I6

). The enzymatic experiments with coniferyl alcohol exhibited the stereochemical diversity, which is in line with the discordance of the predominant enantiomers of secoisolariciresinol isolated from different organs ofA. lappa (Fig. 3).

Pinoresinol/lariciresinol reductase (PLR), responsible for reduction of pinoresinol to lariciresinol, and lariciresinol to secoisolariciresinol, was purified from Forsythia intermediaI4) , and this enzyme was detected from

Zanthoxylum ailanthoidei5)andDaphne odora26). Incubation of (±)-pinoresinols with the seed enzyme preparation yielded almost optically pure (- )-secoisolariciresinol (99% e.e.) and (+)-lariciresinol (85% e.e.), and (-)-secoisolariciresinol (91 % e.e.) was formed from (±)-lariciresinols with the enzyme preparation17). Thus, the seed enzyme preparation had PLR activity. The predominant formation of (- )-secoisolariciresinol from

OH

OH

H

OH

(-)-Secoiso-laricires~ol [38% e.e.] (+ )-Secoiso-lariciresinol [20% e.e.]

HO

(-)-Lariciresinol [>99%c.c.] (+)-Lariciresinol [30%e.e.]

HO

+

OCH3

OH

(-)-Pinoresinol [22% e.e.] HO (+)-Pinoresinol [33% e.e.]

2r

0H H1I1I8'

° ,,:::::,:

I

.

~I"'::"""

0

HOY

OCH3 Petiole

/"

enzyme Coniferyl alcohol

Fig. 3. Formation of pinoresino1, lariciresinol and secoisolariciresinol byArctium lappaenzyme preparations. The petiole enzyme preparation catalyzes the lignan formation in favor of (- )-enantiomer, while the seed enzyme preparation does in favor of (+ )-enantiomer.

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-SUZUKI: Biosynthesis of Lignans and Norlignans

these racemic lignans are in line with the results of incubation of coniferyl alcohol with the seed enzymeI6).

The petiole enzyme also exhibited PLR activity giving rise to lariciresinol and secoisolariciresinol from

(±)-pinoresinols, and secoisolariciresinol from (

±

)-lariciresinols 17). Interestingly, however, the predominant enantiomers of the product lignans, ( - )-secoisola-riciresinol and (+)-lariciresinol, formed from

(±)-pinoresinols and (±)-lariciresinols were the same as those

obtained with the seed enzyme, and ( -

)-secoisolariciresinol is opposite to the predominant enantiomer, (+)-secoisolariciresinol, isolated from the petiole. The enantiomer excess values of the formed (-)-secoisolariciresinol (44 and 37% e.e.) were much lower than those formed with the seed enzyme which are almost optically pure (99 and 91 % e.e.).

These results can be accounted for by postulating thatA.

lappa has PLR isoforms showing different selectivity in terms of the substrates, (+)-pinoresinol and (-)-pinoresinol. Although final conclusions await further experiments, this view is in good accordance with recent findings on PLR of different plant species as follows: PLR was partially purified from Forsythia intermedia cv. Lynwood gold27), and its cDNAs were cloned and expressed in E. coliI4). Both plant and recombinant proteins exhibited the same stereochemical property; each protein catalyzed selective formation of (+)-lariciresinol and (- )-secoisolariciresinol from (±)-pinoresinols, and (- )-secoisolariciresinol from (±)-lariciresinols in the presence ofNADPHI4). PLR of Zanthoxylum ailanthoidei5)

also showed similar stereochemical selectivity to Forsythia PLR. In contrast, PLR activity from Daphne genkwa which exhibited the opposite stereochemical property to the Forsythia and Zanthoxylem PLRs; the Daphne crude enzyme preparation catalyzed selective formation of (-)-lariciresinol (23% e.e.) from (±)-pinoresinols26). These results indicated that different PLRs which have opposite stereochemical properties with respect to lariciresinol and secoisolariciresinol formation distribute in different plant species. Furthermore, the presence of cDNAs cor-responding to the two stereochemically distinct PLRs in a single plant species was demonstrated by Fujita et al.28) although they did not men tion the physiological roles of the two isoforms. On the other hand, the author's present results strongly suggest that the two PLR isoforms are expressed differentially in A. lappa.

As for the stereochemistry of pinoresinol formation, dirigent protein has not yet been isolated from A. lappa. However, a recent detection of a dirigent-protein-like gene from A. lappa using a PCR-guided strategl9) suggests that stereochemistry of formation of pinoresinol from coniferyl alochol in A. lappa is also under control of dirigent protein. In accordance with the presence oflarge amounts of two optically pure dibenzylbutyrolactone lignans, ( - )-matairesinol and ( - )-arctigenin, in A. lappa seeds, secoisolariciresinol dehydrogenase activity was detected in the seed enzyme preparation which gave rise to optically pure (- )-matairesinol following incubation of racemic

(±)-secoisolariciresinols in the presence of NADpI7). Thus, although formation of secoisolariciresinol is controlled stereochemically, the control is not enough strong to produce only one enantiomer of

secoisolari-ciresinol. Formation of optically pure lignan is finally achieved in the conversion of secoisolariciresinol to matairesinol in this plant species.

Taken together, there is a great stereochemical diversity in lignan biosynthesis, and not only the enantioselective coupling of coniferyl alcohol assisted by dirigent protein but also the subsequent several steps must play substantial roles in production of optically pure lignans.

1-2 Stereochemistry of lignan biosynthesis in Anthriscus sylvestris (L.) Hoffm.

Due to the limited supply of podophyllotoxin (Fig. 4) from natural resources5), the alternative sources have been searched. Anthriscus sylvestris (L.) Hoffm. might be one of the candidates, because it produces angeloyl podophyllotoxin30) as well as significant amounts of a precursor of podophyllotoxin, deoxypodophyllotoxin31-36) (Fig. 4). In addition, it produces the typical heartwood lignans yatein and hinokinin34) (Fig. 4), which are found specifically in the heartwood region in the conifers

Libocedrus yateensii7) and Chamaecyparis obtusa38) respectively. Thus, A. sylvestris is probably a good plant material for lignan biosynthesis studies to access mechanisms involved in antitumor and heartwood lignan formation. As the first step, it is necessary to characterize the precursor lignans of yatein and deoxypodophyllotoxin in A. sylvestris.

Suzuki et al.18) preliminarily surveyed lignans in the {3-glucosidase-treated MeOH extracts of both aerial parts and roots of A. sylvestris. GC-MS analysis revealed that the presence of the lignans, yatein and secoisolariciresinol. In addition, nemerosin and deoxypodophyllotoxin, which were previously isolated from Anthriscus Spp.3I--:36,39), were identified by comparing their spectrometoric data 39,40).

After fractionating the f3-glucosidase-treated MeOH extracts of A. sylvestris by silica gel column chromatography, lariciresinol, matairesinol, hinokinin, pluviatolide, and bursehernin (Fig. 4) were identified by GC-MS analysis 18). Secoisolariciresinol, lariciresinol, matairesinol, pulviatolide and brusehernin were identified for the first time in Anthriscus spp.

When coniferyl alcohol was incubated with the A.

sylvestrisenzyme preparation in the presence of H 202 and NADPH, the lignans, pinoresinol, lariciresinol, and secoisolariciresinol, were formed. Furthermore, the enzymatic conversion of (±)-pinoresinol to (+)-lariciresinol (93% e.e.) and (- )-secoisolariciresinol (95% e.e.) by PLR was demonstrated in the presence of NADPH I8 ).

The PLR activity together with enzymatic formation of the lignans from coniferyl alcohol accorded well with those with A. lappa and Forsythia spp6,8,11,27,41,42). In addition, the PLR-catalyzed selective formation of(+)-lariciresinol and (- )-secoisolariciresinol from (±)-pinoresinols with the A. sylvestris enzyme preparation suggested that the stereochemical property of A. sylvestris PLR-catalyzed reduction was similar to those of Forsythia PLR27) and A.

lappa ripening seed PLR 17)

The lignan formation by the Anthriscus enzyme preparation along with the detection of lariciresinol and secoisolariciresinol from the plant suggests strongly that the conversion, pinoresinollariciresinolsecoisolaricire55

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-Lariciresinol

OOh

Secoiso-lariciresinol OH Matairesinol OH Rl=OCH3, R2=CH3, Yatein Rl=H, R2=H, Pluviatolide -Rl=H, R2=CH3, Bursehemin Hinokinin Nemerosin

o

Deoxypodo-phyllotoxiri Podophyllotoxin

Fig. 4. Chemical structures of podophyllotoxin and Anthriscus sylvestris lignans.

attention has been paid to regulating the norlignan biosynthesis.

Several hypothetical pathways for norlignan biosyn-Asparenydiol: R1=H, R2=H

Asparenyol: Rl=H, Ri=CH3 Asparenyn:R1

=

CH3,R2=CH3

Fig. 5. Chemical structures of norlignans and related compounds.

H~OH

(E)-Hinokiresinol OH OH OH Sequirin-C

RI0~O-o-OR2

OH Agatharesinol HO

Chapter II Establishment of norlignan biosynthetic patit.way

Typical norlignans having the I, 3-diphenylpentane [C6-C3(C 2)-C6] structure le.g. hinokiresinol [(E)-hino-kiresinol], agatharesinol, and sequirin-C, Fig. 51 occur in coniferous trees (especially in heartwood) of Cupressaceae, Taxodiaceae, and Araucariaceae43-46), while y-lactonized 1, 3..;diphenylpentane norlignans (e.g. pueroside A and B) were isolated from two Leguminosae trees (Pueraria lobata and Sophora japonica)47-49). Some monocotyledonous Liliaceae and H ypoxidaceae plants are also good sources of 1,3-diphenylpentane and 1,5-diphenylpentane norli-gnans. For instance, (Z)-hinokiresinol (=nyasol) (Fig. 5) which is the geometrical isomer of a coniferous heartwood norlignan, (E)-hinokiresinol, was isolated from Asparagus and Anemarrhena3,50,51).

Itis well known that norlignans accumulate specifically in conifer heartwood. Heartwood coloration ofC.japonica (Japanese cedar) 52,53) and Chamaecyparis obtusa (hinoki cypress) 54) is due to norlignans. The- normal heartwood coloration ofC. japonicaand C. obtusais pale salmon pink, which has been appreciated in Japan. However, black-discoloration often occurs in C. japonicaheartwood, which lowers the valueofthe wood. To solve the problem, much sinol.

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SUZUKI: Biosynthesis of Lignans and Norlignans

OH

HO

6&,O'I':OH

[l3C]Cinnamic 5~ 3 4 acids (Z)-Hinokiresinol

Fig. 6. 13C-Labelling patterns of (Z)-hinokiresinol incorporating [7- 13C]cinnamic acid, [8_ 13C] cinnamic acid, or[9-13C]cinnamic acid.

....=

7-13C;

.=8_

13C;

.=9_

13C.

individ ually, and the 13C-enriched position III the side

chain of (Z)-hinokiresinol was determined by GC-MS and 13C NMR. When [7-13C]cinnamic acid was adminis-tered, specific 13C enrichments at C-l (11.7 atom% excess) and C-3 (10.6 atom% excess) of (Z)-hinokiresinol were observed. Similarly,13C enrichments at C-2 (32.3 atom% excess) and C-4 (31.2 atom% excess) occurred when [8-13C ]cinnamic acid was fed. As for the feeding of [9_13C] cinnamic acid, significant 13C enrichment at only C-5 (26.3 atom% excess) was observed. 13C enrichments at other positions were negligible (-0.27-0.56 atom% excess).

These 13C-tracer experiments unequivocally established that all 17 carbon atoms of (Z)-hinokiresinol are derived from phenylpropanoid monomers. Also, it was conclusively demonstrated that the side chain, 7-C, 8-C, and 9-C atoms of cinnamic acid were incorporated into C-l and C-3, C-2 and C-4, and C-5 of (Z)-hinokiresinol, respectively (Fig. 6). Thus, intramolecular rearrange-ment of the side chain carbon atoms of the monomers did not occur in (Z)-hinokiresinol formation.

Suzuki et a/.61) next demonstrated the immediate

precursor(s) in (Z)-hinokiresinol formation. First, they synthesized the following 13C and/or 2H labelled compounds, 4-[ring-13C6]coumaric acid, 4-[9, 9-2H2, ring-13C6]coumaryl alcohol, 4-[7,9, 9-2H 3]coumaryl alcohol, and 4-[9- 2H , ring-13C 6]coumaraldehyde, and then administered the compounds individually to the elicited

Asparagus cells.

When 4-[ring-13C6]coumaric acid was fed, GC-MS

analysis of the formed (Z)-hinokiresinol showed the significant enhancement of ion peak at m/z 408

([M]+

+

12), indicating that 4-coumaric acid was on the metabolic pathway leading to (Z)-hinokiresinol. When 4-[9,9-2H2 , ring-13C6]coumaryl alcohol was fed to the cells,

great enhancement of ion peak at m/z410 was observed. This result demonstrated that two units of 4-coumaryl alcohol were converted ultimately to (Z)-hinokiresinol with the loss of the two 9-positioned deuterium atoms from one of the monomers, but did not imply that two units of the alcohol were directly involved in dimerization giving rise to

(Z)-hinokiresinol.

Importantly, when 4-[9,9-2H 2, ring-I3C6] coumaryl alcohol was administered, enhancement at m/z 404

([M]+

+

8) was also observed, which was assigned to

(Z)-[2H2 , 13C6]hinokiresinol TMS ether, i.e. the product of coupling of one unit of exogenous 4-[9,9-2H 2, ring-13

C6 ] coumaryl alcohol with an endogenous unlabelled phenylpropane unit. This endogenous precursor-induced dilution effect is rather common in feeding experiments, and, in fact, also occurred in the case of L-[ring_13C6]

phenylalanine administration. In addition to the II-I Pathway of norlignan biosynthesis

Within the last decade, Takasugi63) reported that a herbaceous plant, Asparagus officina/is, inoculated with a

phytopathogen produced (Z)-hinokiresinol (Fig. 5) as a phytoalexin. Terada et a/.64) reported that cell cultures of the plant produced norlignan-related C 6-C5-O-C6 compounds, asparenydiol and its methylated compounds (asparenyol and asparenyn) (Fig. 5), without any elicitor treatment. Later, they demonstrated that asparenyol was derived from two units of phenylalanine with a loss of one carbon atom at the 9-position of phenylalanine based onl3C tracer experiments65), and assumed hinokiresinol as a putative precursor of asparenyol, although without any experimental evidence66).

Suzuki et a/.61) established a A. officinalis cell system

producing a norlignan, (Z)-hinokiresinol (yield: 0.02% based on dried cell weight). They isolated

(Z)-hinokiresinol from MeOH extract of elicitor-treated A.

officina/is cells and identified it by NMR analysis.

Next, L-[ring- 13C6] phenylalanine was administered to

the elicitor-treatedA. officina/is cells, and the

fi-glucosidase-treated MeOH extract was submitted to GC-MS analysis to examine the incorporation of 13C . Compared with the mass spectrum of unlabelled (Z)-hinokiresinol TMS ether

([M]+=m/z 396), the enhanced ion peak at m/z 408

([M]+

+

12) was observed, indicating unequivocally that two aromatic rings of (Z)-hinokiresinol were derived from L-phenylalanine.

Similarly, cinnamic acids labelled with 13C at the side chain were next administered to the Asparagus cells

thesis had been proposed based on the chemical structures of norlignans37,43,55--60). First, Enzell and Thomas58) suggested the coupling of two phenylpropane units followed by a loss of one carbon atom giving rise to agatharesinol. Later, a coupling of 4-coumaric acid with 4-coumaryl alcohol that involved the loss of the carbon atom at the 9-position of 4-coumaric acid was proposed independently by Birch and Liepa59), and Beracierta and Whiting60) Erdtman and Harmatha37) subsequently assumed that C8-C8' linked lignans formed by the coupling of two phenylpropanoid monomers were converted to norlignans via an intramolecular rearrangement of the side chain of the carbon skeleton. Despite these proposals of coupling modes of two phenyl propane units, none of them were supported by any concrete experimental evidence. In this chapter, the establishment of norlignan biosynthetic pathway is described. Using a fungal-elicited Asparagus officina/is cell system, it has been demonstrated that (Z)-hinokiresinol originates from two non-identical phenylpropanoid monomer: 4-coumaryl alcohol and a 4-coumaroyl compound61). Furthermore, the first in vitro norlignan formation with an enzyme

preparation has been demonstrated62). The enzyme preparation from fungal-elicitedA. officina/is cells catalyzed

the formation of (Z)-hinokiresinol from two non-identical phenylpropanoid monomers, coumaryl alcohol and 4-coumaroyl CoA, and from a dimer, 4-coumaryl 4-coumarate, without any additional cofactors. Based on the results of the enzymatic reaction, the novel biosyn thetic mechanism for (Z)-hinokiresinol via the ester enolate Claisen rearrangement is proposed.

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(Z)-Hinokiresinol Fig. 7. Proposed biosynthetic pathway for

(Z)-hinokiresinol. The C6-C3 moiety of (Z)-hinokiresinol was derived from 4-coumaryl alcohol, while the C6-C2 moiety was from a

4-coumaroyl compound (4-coumaric acid, R OH; 4-coumaroyl CoA, R=SCoA ; 4-coumaral-dehyde, R=H).

from phenylpropanoid monomers with the loss of one carbon atom at the 9-position of one of the monomers. TheC6-C3 moiety of (Z)-hinokiresinol is originated from 4-coumaryl alcohol, while. the C 6-C2 moiety is from a 4-coumaroyl compound.

11-2 First enzymatic formation of the norlignan Suzuki et al.62) demonstrated in vitro norlignan formation for the first time. Thus, an enzyme preparation from fungal-elicited Asparagus officinalis cultured cells catalyzed the formation of a norlignan, (Z)-hinokiresinol, from two non-identical phenylpropanoid monomers, 4-coumaryl alcohol and 4-coumaroyl CoA, and from a dimer, 4-coumaryI4-coumarate, without any additional cofactors. Proof that the formation of (Z)-hinokiresinol was enzymatic was obtained by control experiments; the formation of (Z)-[2H 3]hinokiresinol from 4-[7, 9, 9-2H3] coumaryl alcohol and 4-coumaroyl CoA did not occur when the denatured enzyme preparation was used, and barely occurred when the enzyme preparation or the substrate(s) were omitted from the complete assay. On the other hand, incubation of 4- [7, 9, 9-2H3] coumaryl alcohol and unlabelled 4-coumarate with the enzyme preparation did not afford (Z)-[2H3]hinokiresinol, eliminating the mechanism that 4-coumaroyl CoA was first hydrolyzed to 4-coumarate, which coupled with 4-coumaryl alcohol to afford (Z)-hinokiresinol. These results demonstrate for the first time a norlignan synthase activity.

Since esters are often biosynthesised by acyltransferase-catalyzed condensation between the corresponding CoA esters and alcohoI70), Suzukiet al.62)next hypothesized that

(Z)-hinokiresinol was formed via the ,coupling of coumaryl alcohol and coumaroyl CoA to afford 4-coumaroyl 4-coumarate followed by C7-C8'·, bond formation and C9' decarboxylation (Fig. 8). To test this hypothesis, 4-[7,9, 9-2H 3]coumaryl 4-coumarate was

~

OH '?' . .

~I

OH 4-Coumaryl alcohol

--R 9 0 Cinnamic acid L-Phenyl-alanine significant enhancement of the ion peak at m/z 408

([M]+ + 12); due to the incorporation of two

e

3C 6] phenylalanine units into (Z)-hinokiresinol, great enhancement was also observed at m/z 402 ([M] ++6), and may be ascribed to coupling of one

e

3C 6]phenylalanine unit and one endogenous unlabelled phenylpropane unit. Interestingly, however, the ion peak atm/z402 ([M]++6, (Z)-[13C6]hinokiresinol TMS ether) after 4-[9, 9-2H 2, ring-13C6]coumaryl alcohol' administration was not significant. If one such labelled 4-coumaryl alcohol unit and one endogenous unlabelled 4-coumaryl alcohol unit are directly involved in the dimerization, both [M] + + 8 and [M] + + 6 ions must appear with equal intensity. This suggests that two 4-coumaryl alcohol units were not involved directly in coupling, and that the coupling of one 4-coumaryl alcohol unit and another phenylpropane' unit which can be formed from 4-coumaryl alcohol.

Itis established that the reduction of cinnamaldehyde and cinnan;l:Oyl CoA by cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl CoA reductase (CCR), respectively, is reversible67-69). Hence, it was hypothesized that some of the exogenously administered 4-[9, 9-2H2, ring_13C6 ]

coumaryl alcohol were converted to 4-[9-2H , ring_13C6 ] coumaraldehyde and 4-[ring_13C6 ]coumaroyl CoA, which

in turn coupled with 4-[9, 9-2H2, ring_13C6]coumaryl alcohol to afford (Z)-[2H2, 13C12]hinokiresinol.

To test this hypothesis, the simultaneous administration of two distinct, possible precursors was carried out61). Thus, equal molar amounts of4-[ring_13C6]coumaricacid

and 4-[7,9, 9-2H 3]coumaryl alcohol were administered to elicited cells in a single flask, and the results were compared with those obtained after individual administration of the two precursors as positive controls. Administration of 4-[7,9,9-2H3]coumaryl alcohol alone resulted in formation of (Z)-eH4]hinokiresinol TMS ether

([M]+ +4) and (Z)-eH 3]hinokiresinol TMS ether ([M]+ +3) which corresponded to (Z)-eH2, 13C12] hinokiresinol TMS ether ([M]+ + 14) and (Z)-eH2, 13C6] hinokiresinol TMS ether ([M]+ +8), respectively, in the 4-[9, 9-2H2, ring_13C6]coumaryl alcohol administration. Similarly, administration of only 4-[ring-13C6]coumaric acid resulted in the enhanced ion peaks of [M] + + 12

I

(Z)-e

3

C 12]hinokiresinol TMS etherl as already described. In sharp contrast, the simultaneous administration of the two precursors provided no significant evidence in coupling products of two units of 4-[7,9, 9-2H 3]coumaryl alcohol ([M]+ +4, (Z)-eH4]hinokiresinol TMS ether).

In addition, the ion peak at m/z 408 ([M]+ + 12,

(Z)-[13C 12] hinokiresinol TMS ether) showed only a small increase, compared with the unlabelled one. The ion peak at m/z 405 ([M]++9) was prominent, and was derived by coupling one 4-[7,9, 9-2H 3]coumaryl alcohol unit with one4-[ring_13C6]coumaricacid unit, confirming

our hypothesis that (Z)-hinokiresinol is not formed by the direct dimerization of two units of 4-coumaryl alcohol. Instead, the C6-C3' moiety of (Z)-hinokjresinol, is derived from 4-coumaryl alcohol unit, while the C 6-C 2 moiety is from a 4-coumaroyl compound (HO-C6H5-CH=CH-CO-R) such as 4-coumaric acid, 4-coumaroyl CoA, or 4-coumaraldehyde .(Fig. 7).

In conclusion, it has been shown for the first time that all carbon atoms ofa norlignan, (Z)-hinokiresinol, are derived

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SUZUKI: Biosynthesis of Lignans and Norlignans

Conclusions

(Z)-Hinokiresinol

Fig. 8. A putative mechanism for the formation of

(2)-hinokiresinol with A. officinalis enzyme

prepara-tion. Acknowledgement

The author wishes to thank Associate Professor Dr. Toshiaki Umezawa, Wood Research Institute, Kyoto University, for many helpful discussions and critical reading of the manuscript.

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In conclusion, the present study has demonstrated for the first time the enzymatic formation of (Z)-hinokiresinol from 4-coumaryl alcohol and 4-coumaroyl CoA, and from 4-coumaryl 4-coumarate.

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